HPYY(1-36) Having a Beta-Homoarginine Substitution at Position 35

ABSTRACT

The invention relates to PYY compounds having the amino acid in the position corresponding to position 35 of hPYY(1-36) substituted with beta-homoarginine and derivatives thereof with a modifying group attached to the position corresponding to position 7 of hPYY(1-36). The compounds of the invention are selective Y2 receptor agonists. The invention also relates to pharmaceutical compositions comprising such PYY compounds and pharmaceutically acceptable excipients, as well as the medical use of the PYY compounds.

TECHNICAL FIELD

The present invention relates to analogues and/or derivatives of PeptideYY (PYY), and their pharmaceutical use.

INCORPORATION-BY-REFERENCE OF THE SEQUENCE LISTING

The Sequence Listing, entitled “SEQUENCE LISTING”, is 25054 bytes, wascreated on 12 Nov. 2013 and is incorporated herein by reference.

BACKGROUND OF THE INVENTION

PYY is released during a meal from L-cells in the distal small intestineand the colon. PYY is known to have peripheral effects in thegastrointestinal (GI) tract and also act centrally as a satiety signal.PYY is naturally secreted as a 36 amino acid peptide (PYY(1-36)) with aC-terminal amide but is cleaved to PYY(3-36) which constitutesapproximately 50% of the circulating PYY. The enzyme responsible for thedegradation is dipeptidyl peptidase IV (DPPIV). PYY(3-36) is rapidlyeliminated by proteases and other clearance mechanisms. The half-life ofPYY(3-36) has been reported to be <30 minutes in pigs (Ito T et al,Journal of Endocrinology (2006), 191, pp113-119). Thus, PYY displayssuboptimal pharmacokinetic properties, meaning that the peptide has tobe administered at least twice daily.

Whereas PYY(1-36) activates Y1, Y2 and Y5 receptors with very littleselectivity and the Y4 receptor slightly less, the DPP IV processedPYY(3-36) displays increased selectivity for the Y2 receptor over Y1, Y4and Y5 receptors, albeit some Y1 and Y5 affinity is retained. Y2receptor activation is known to decrease appetite and food intakewhereas Y1 and Y5 receptor activation leads to an increase in appetiteand food intake. Furthermore, Y1 and Y5 receptor activation may lead toan increase in blood pressure.

PYY(3-36) has been suggested for use in the treatment of obesity andassociated diseases based on the demonstrated effects of certain ofthese peptides in animal models and in man, and on the fact that obesepeople have low basal levels of PYY as well as lower meal responses ofthis peptide. Furthermore, Y2 agonists have been demonstrated to haveanti-secretory and pro-absorptive effects in the gastro-intestinal (GI)tract. The potential use of Y2 agonists in the treatment of a number ofgastro-intestinal disorders has been suggested.

Based on demonstrated effects in e.g. Zucker rats and diet induced obese(DIO) mice Y2 selective PYY(3-36) analogues have a positive effect onglucose metabolism and are thus suggested to be used for the treatmentof diabetes (van den Hoek A. et al., Am J Physiol Endocrinol Metab(2006), 292, ppE238-E245; and Ortiz A. et al, The Journal ofPharmacology and Experimental Therapeutics (2007), 323, pp 692-700).

WO 2009/138511 A1 relates to long-acting Y2 and/or Y4 receptor agonists.WO 2011/033068 A1 relates to PYY analogues stabilised against C-terminalproteolytic breakdown. WO 2011/058165 A1 relates to Y2 receptor agonistswith protracted pharmacokinetic properties.

For the treatment of conditions responsive to Y receptor modulation suchas obesity and diabetes it would be attractive to use PYY analogueswhich are specific for the Y receptor subtype Y2 and importantly alsodisplay protracted pharmacokinetic properties and as such can be used ina dosing regimen with lower frequency of administration than PYY orPYY(3-36).

SUMMARY

The invention relates to PYY compounds comprising beta-homoarginine inthe position corresponding to position 35 of human PYY(1-36)(hPYY(1-36), SEQ ID NO:1).

Also or alternatively, in one aspect, the PYY compounds further comprisea lysine in the position corresponding to position 7 of hPYY(1-36), anda modifying group attached to the epsilon amino group of this lysine.

In one aspect, the invention also relates to pharmaceutical compositionscomprising such PYY compounds and pharmaceutically acceptableexcipients, as well as the medical use of the PYY compounds.

Also or alternatively, in one aspect, the invention relates to PYYcompounds being Y2 receptor agonists.

Also or alternatively, in one aspect, the invention relates to PYYcompounds displaying selectivity towards the Y receptor subtype Y2 ascompared to Y receptor subtypes Y1, Y4 and Y5.

Also or alternatively, in one aspect, the invention relates to PYYcompounds with longer half-life than the half-life of hPYY(3-36). Alsoor alternatively, in one aspect, the invention relates to PYY compoundswith longer half-life than the half-life of hPYY(1-36).

DESCRIPTION OF THE INVENTION

The invention relates to PYY compounds. The PYY compounds of the presentinvention have the amino acid in the position that corresponds toposition 35 of hPYY(1-36) substituted with beta-homoarginine.

Also or alternatively, in one aspect, the PYY compounds further comprisea lysine in the position corresponding to position 7 of hPYY(1-36), anda modifying group attached to the epsilon amino group of this lysine.

Also or alternatively, in one aspect, the invention relates to PYYcompounds being Y receptor subtype Y2 agonists.

Also or alternatively, in one aspect, the invention relates to PYYcompounds displaying selectivity towards the Y receptor subtype Y2 ascompared to Y receptor subtypes Y1, Y4 and Y5.

In one aspect peptides being “selective” for specific receptors overother receptors refers to peptides that display at least 10 fold, suchas at least 20 fold, at least 50 fold, or at least 100 fold higherpotency for one Y receptor over other Y receptors as measured in vitroin an assay for receptor function, such as an Actone functional potencyassay, and compared by EC50 values, or a Scintillation Proximity Assay(SPA) measuring receptor binding affinity, and compared by Ki values.

In what follows, Greek letters may be represented by their symbol or thecorresponding written name, for example: α=alpha; β=beta; ε=epsilon;γ=gamma; ω=omega; etc.

PYY Compounds

The term “hPYY(1-36)” as used herein refers to the human Peptide YY, thesequence of which is included in the sequence listing as SEQ ID NO:1.The peptide having the sequence of SEQ ID NO:1 may also be designatednative hPYY.

The term “PYY compound” as used herein refers to a peptide, or acompound, which is a variant of hPYY(1-36). The term “PYY compound” asused herein may also refer to a peptide, or a compound, which is avariant of hPYY(3-36) (SEQ ID NO:2).

The term “PYY compound” as used herein may also refer to a peptide, or acompound, which is a variant of hPYY(4-36).

The C-terminal of the PYY compounds of the present invention is anamide, as is the C-terminal of native hPYY(1-36) (SEQ ID NO:1) andhPYY(3-36) (SEQ ID NO:2), respectively.

The PYY compounds of the present invention can be PYY analogues and/orderivatives thereof.

The term “PYY analogue” is used for PYY compounds, where at least oneamino acid modification in the backbone is present.

The term “PYY derivative” is used for PYY compounds comprising at leastone non-amino acid substituent covalently attached.

A derivative of a PYY analogue is thus a PYY compound comprising atleast one amino acid modification and at least one non-amino acidsubstituent covalently attached.

The PYY compounds of the present invention may comprise up to 10 aminoacid modifications as compared to hPYY(3-36).

The term “amino acid modification” used throughout this application isused in the meaning of a modification to an amino acid as compared tohPYY(3-36). This modification can be the result of a deletion of anamino acid, addition of an amino acid, substitution of one amino acidwith another or a substituent covalently attached to an amino acid ofthe peptide.

The PYY compounds of the invention comprises L-beta-homoarginine(beta-homoArg) at the position corresponding to position 35 ofhPYY(1-36), meaning that the PYY compounds of the invention may compriseup to 9 amino acid modifications in addition to this modification in theposition corresponding to position 35 of hPYY(1-36).

In yet another aspect, the PYY peptides of the invention may exhibit atleast 70%, 75% or 80% sequence identity to hPYY(3-36). As an example ofa method for determination of the sequence identity between twoanalogues the two peptides [beta-homoArg35]hPYY(3-36) and hPYY(3-36) arealigned. The sequence identity of the [beta-homoArg35]hPYY(3-36)analogue relative to hPYY(3-36) is given by the number of alignedidentical residues minus the number of different residues divided by thetotal number of residues in hPYY(3-36). Accordingly, in said example thesequence identity is (34-1)/34.

PYY compounds or PYY analogues of the invention may be described byreference to i) the number of the amino acid residue in hPYY(1-36) whichcorresponds to the amino acid residue which is changed (i.e., thecorresponding position in hPYY(1-36), and to ii) the actual change.

The following is a non-limiting example of suitable analoguenomenclature. [beta-homoArg35]hPYY(3-36) designates an analogue of thehuman PYY(1-36), wherein the naturally occurring arginine in position 35has been substituted with L-beta-homoarginine and the naturallyoccurring tyrosine and proline in position 1 and 2, respectively, havebeen deleted.

The following is a non-limiting example of suitable analoguenomenclature. [beta-homoArg35]hPYY(3-36) designates an analogue of thehuman PYY(1-36), wherein the naturally occurring arginine in position 35has been substituted beta-homoarginine and the naturally occurringtyrosine and proline in position 1 and 2, respectively, has beendeleted.

The following is a non-limiting example of suitable nomenclature for aderivative of a PYY analogue.7-N{Epsilon}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]-ethoxy]acetyl]-[Lys7,beta-homoArg35]hPYY(3-36)designates a derivative of an analogue of the human PYY(3-36), wherein[Lys7,beta-homoArg35] designate the amino acid changes as compared tohPYY(3-36) (SEQ ID NO:2), and wherein the substituent[2-[2-[2[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]-amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-is attached to the epsilon amino group of the lysine in the positioncorresponding to position 7 in hPYY(1-36).

The expressions “a position equivalent to” or “corresponding position”may be used to characterise the site of change in a variant PYY sequenceby reference to hPYY(1-36).

In general throughout the application, when referring to a particularposition of a PYY analogue, the position referred to is the position ofthe PYY analogue corresponding to that particular position ofhPYY(1-36).

The expression used throughout this application, that a PYY compoundcomprises a particular amino acid at a position corresponding to acertain position of hPYY(1-36), means that the native amino acid in thatposition has been replaced with that particular amino acid.

Amino acid residues may be identified by their full name, theirone-letter code, and/or their three-letter code. These three ways arefully equivalent.

Analogues “comprising” certain specified changes may comprise furtherchanges, when compared to hPYY(1-36). In one aspect, the analogue “has”the specified changes.

PYY Analogues

A PYY analogue is a PYY peptide in which a number of amino acid residueshave been modified when compared to hPYY(1-36). These modificationsinclude substitutions, insertions, and/or deletions, alone or incombination.

In a specific aspect, the PYY analogues of the invention include one ormore modifications of a “non-essential” amino acid residue. In thecontext of the invention, a “non-essential” amino acid residue is aresidue that can be altered, i.e., deleted or substituted in the humanPYY amino acid sequence without abolishing or substantially reducing theactivity of the PYY analogue towards the Y2 receptor.

Substitutions.

In one aspect amino acids may be substituted by conservativesubstitution. The term “conservative substitution” as used hereindenotes that one or more amino acids are replaced by another,biologically similar residue. Examples include substitution of aminoacid residues with similar characteristics, e.g. small amino acids,acidic amino acids, polar amino acids, basic amino acids, hydrophobicamino acids and aromatic amino acids.

In one aspect, the PYY analogues of the invention may comprisesubstitutions of one or more unnatural and/or non-amino acids, e.g.,amino acid mimetics, into the sequence of PYY.

Deletions and Truncations.

In one aspect, the PYY analogues of the invention may have one or moreamino acid residues deleted from the amino acid sequence of human PYY,alone or in combination with one or more insertions or substitutions.

Insertions.

In one aspect, the PYY analogues of the invention may have one or moreamino acid residues inserted into the amino acid sequence of human PYY,alone or in combination with one or more deletions and/or substitutions.

In one aspect, the PYY analogues of the invention may include insertionsof one or more unnatural amino acids and/or non-amino acids into thesequence of PYY.

The PYY peptide may be derived from vertebrates, such as human, mouse,sheep, goat, cow, or horse. The term “vertebrate” means members of thesubphylum Vertebrata, a primary division of the phylum Chordata thatincludes the fish, amphibians, reptiles, birds, and mammals, all ofwhich are characterized by a segmented spinal column and a distinctwell-differentiated head. The term “mammal” means humans as well as allother warm-blooded members of the animal kingdom possessed of ahomeostatic mechanism in the class Mammalia, e.g., companion mammals,zoo mammals, and food-source mammals. Some examples of companion mammalsare canines (e.g., dogs), felines (e.g., cats) and horses; some examplesof food-source mammals are pigs, cattle, sheep, and the like. In oneaspect the mammal is a human or a companion mammal. In one aspect themammal is a human, male or female.

The term “peptide”, as e.g. used in the context of the PYY compounds ofthe invention, refers to a compound which comprises a series of aminoacids interconnected by amide (or peptide) bonds.

The PYY peptides of the invention comprise at least 25 constituent aminoacids connected by peptide bonds. In particular embodiments the PYYpeptides comprise at least 33 amino acids. In particular embodiments thePYY peptides comprise at least 34 amino acids.

Amino acids are molecules containing an amine group and a carboxylicacid group, and, optionally, one or more additional groups, oftenreferred to as a side chain.

The term “amino acid” includes proteinogenic (or natural) amino acids(amongst the 20 standard amino acids), as well as non-proteinogenic (ornon-natural) amino acids. Proteinogenic amino acids are those which arenaturally incorporated into proteins. The standard amino acids are thoseencoded by the genetic code. Non-proteinogenic amino acids are eithernot found in proteins, or not produced by standard cellular machinery(e.g., they may have been subject to post-translational modification).Non-limiting examples of non-proteinogenic amino acids are Aib(α-aminoisobutyric acid), beta-homoArg (L-beta-homoarginine), as well asthe D-isomers of the proteinogenic amino acids.

In what follows, all amino acids of the PYY compound for which theoptical isomer is not stated is to be understood to mean the L-isomer(unless otherwise specified).

PYY Derivatives

The term “derivative” as used herein in the context of a PYY peptide oranalogue means a chemically modified PYY peptide, in which one or moresubstituents have been covalently attached to the peptide.

In one aspect of the invention, the substituent may be an N-terminalsubstituent.

Also or alternatively, in one aspect, the substituent may be a modifyinggroup or alternatively, referred to as a protracting moiety.

N-Terminal Substituents

In one aspect of the invention, the PYY compound comprises a substituentcovalently attached to the alpha-amino group in the amino acid residuein the N-terminus of the PYY compound. In one aspect, the amino acidresidues in the positions corresponding to positions 1-3 of hPYY(1-36)are absent, and the N-terminal substituent is covalently attached to theamino acid residue in the position corresponding to position 4 ofhPYY(1-36).

In one aspect, the N-terminal substituent is an alkoxy group. In oneaspect, the N-terminal substituent is an alkoxy group comprising up to12 carbon atoms. In another aspect, the N-terminal substituent is analkoxy group comprising up to 6 carbon atoms.

Modifying Group/ Protracting Moiety

In one aspect, the PYY compound comprises a substituent or modifyinggroup covalently attached to the amino acid residue in the positioncorresponding to position 7 of hPYY(1-36). In one further aspect, thesubstituent or modifying group is capable of forming non-covalentconjugates with proteins, thereby promoting the circulation of thederivative with the blood stream, and also having the effect ofprotracting the time of action of the derivative, due to the fact thatthe conjugate of the PYY derivative and albumin is only slowly removedby renal clearance. Thus, the substituent, or modifying group, as awhole may also be referred to as a protracting moiety.

The modifying group may be covalently attached to a lysine residue ofthe PYY peptide by acylation, i.e., via an amide bond formed between acarboxylic acid group of the modifying group and the epsilon amino groupof the lysine residue. The amino group of lysine could also be coupledto an aldehyde of the modifying group by reductive amination. In anotheraspect the thiol group of cysteine could by coupled to a maleiimidogroup of the modifying group by Michael addition or coupled to thechloro- or iodoacetyl group of the modifying group by nucleophilicsubstitution.

In one aspect, the modifying group may be covalently attached to alysine residue in a position corresponding to position 7 of hPYY(1-36)by acylation, i.e., via an amide bond formed between a carboxylic acidgroup of the modifying group and the epsilon amino group of the lysineresidue.

The derivatives of the invention may exist in different stereoisomericforms having the same molecular formula and sequence of bonded atoms,but differing only in the three-dimensional orientation of their atomsin space. The stereoisomerism of the exemplified derivatives of theinvention is indicated in the experimental section, in the names as wellas the structures, using standard nomenclature. Unless otherwise statedthe invention relates to all stereoisomeric forms of the claimedderivative.

In one aspect of the invention, all amino acids of the PYY compound forwhich the optical isomer is not stated is to be understood to mean theL-isomer (unless otherwise specified).

Pharmaceutically Acceptable Salts

The PYY compounds of the invention may be in the form of apharmaceutically acceptable salt.

Salts are e.g. formed by a chemical reaction between a base and an acid,e.g.: 2NH₃+H₂SO₄→(NH₄)₂SO₄.

The salt may be a basic salt, an acidic salt, or it may be neither nor(i.e. a neutral salt). Basic salts produce hydroxide ions and acid saltshydronium ions in water.

The salts of the derivatives of the invention may be formed with addedcations or anions between anionic or cationic groups, respectively.These groups may be situated in the peptide moiety, and/or in the sidechain of the derivatives of the invention.

Non-limiting examples of anionic groups of the derivatives of theinvention include free carboxylic groups in the side chain, if any, aswell as in the peptide moiety. The peptide moiety often includes freecarboxylic groups at internal acid amino acid residues such as Asp andGlu.

Non-limiting examples of cationic groups in the peptide moiety includethe free amino group at the N-terminus, if present, as well as any freeamino group of internal basic amino acid residues such as His, Arg, andLys.

Functional Properties

In a first functional aspect, the PYY compounds of the invention have agood Y2 receptor potency. Also, or alternatively, in a second aspect,they bind very well to the Y2 receptor. Preferably they are full Y2receptor agonists as is reflected by their ability to bind strongly tothe Y2 receptor combined with the capacity to fully activate thereceptor compared to hPYY(1-36) and hPYY(3-36).

Also or alternatively, in a second functional aspect, the inventionrelates to PYY compounds displaying selectivity towards the Y receptorsubtype Y2 as compared to Y receptor subtypes Y1, Y4 and Y5.

Also, or alternatively, in a third functional aspect, the PYY compoundsof the invention have improved pharmacokinetic properties. Also, oralternatively, in a fourth functional aspect, the PYY compounds of theinvention have increased half-life and/or a decreased clearance. Also,or alternatively, in a fifth functional aspect, they have the effect invivo of decreasing the blood glucose. Also, or alternatively, in a sixthfunctional aspect, they have the effect in vivo of decreasing foodintake.

Biological Activity—In Vitro Potency

According to the first functional aspect, the PYY compounds of theinvention are biologically active, or potent.

In a particular embodiment, potency and/or activity refers to in vitropotency, i.e. performance in a functional Y2 receptor assay, more inparticular to the capability of activating the human Y2 receptor.

The term half maximal effective concentration (EC₅₀) generally refers tothe concentration which induces a response halfway between the baselineand maximum, by reference to the dose response curve. EC₅₀ is used as ameasure of the potency of a compound and represents the concentrationwhere 50% of its maximal effect is observed.

The in vitro potency of the derivatives of the invention may bedetermined as described in Example 31, and the EC₅₀ of the derivative inquestion determined. The lower the EC₅₀ value, the better the potency.

In one aspect, the derivative of the invention has an in vitro potencydetermined using the method of Example 31 corresponding to an EC₅₀ at orbelow 100 nM. In another aspect, the derivative of the invention has anin vitro potency determined using the method of Example 31 correspondingto an EC₅₀ at or below 50 nM. In another aspect, the derivative of theinvention has an in vitro potency determined using the method of Example31 corresponding to an EC₅₀ at or below 25 nM.

Biological Activity—In Vitro Receptor Binding

According to the second functional aspect, the PYY compounds of theinvention bind very well to the Y2 receptor. This may be determined asdescribed in Example 32.

Generally, the binding to the Y2 receptor should be as good as possible,corresponding to a low Ki value. The Ki value is determined by theCheng-Prusoff equation Ki=IC50/(1+[L]/Kd), wherein IC50 is the halfmaximal inhibitory concentration of the agonist, [L] is theconcentration of the radioligand and Kd is the dissociation constant forbinding.

As an example, in one aspect, the Y2 receptor binding affinity (Ki) isbelow 100 nM. In one aspect, the Y2 receptor binding affinity (Ki) isbelow 50 nM. In one aspect, the Y2 receptor binding affinity (Ki) isbelow 10 nM.

Biological Activity—In Vivo Pharmacology

In another particular embodiment the PYY compounds of the invention arepotent in vivo, which may be determined as is known in the art in anysuitable animal model, as well as in clinical trials.

The diabetic db/db mouse is one example of a suitable animal model, andthe blood glucose lowering effect may be determined in such mice invivo, e.g. as described in Example 34.

In addition, inhibition of food intake in the db/db mice is a suitablemodel for determination of effect on food intake and body weight as alsodescribed in Example 34.

Generally, the glucose lowering effect of a 1 umol/kg dose should be asgood as possible corresponding to a low relative % glucose level.

As an example, in a particular aspect of the invention, 16 hours afterdosing the relative % glucose level is below 80%. In one aspect of theinvention, 16 hours after dosing the relative % glucose level is below70%. aspect of the invention, 16 hours after dosing the relative %glucose level is below 60%.

As an example, in a particular aspect of the invention, 16 hours afterdosing the % relative food intake is below 40%. In one aspect of theinvention, 16 hours after dosing the % relative food intake is below30%. In one aspect of the invention, 16 hours after dosing the %relative food intake is below 20%.

Pharmacokinetics Profile

According to the third functional aspect, the PYY compounds of theinvention have improved pharmacokinetic properties such as increasedterminal half-life and/or decreased clearance.

Increasing terminal half-life and/or decreasing of the clearance meansthat the compound in question is eliminated slower from the body. Forthe compounds of the invention this entails an extended duration ofpharmacological effect.

The pharmacokinetic properties of the derivatives of the invention maysuitably be determined in-vivo in pharmacokinetic (PK) studies. Suchstudies are conducted to evaluate how pharmaceutical compounds areabsorbed, distributed, and eliminated in the body, and how theseprocesses affect the concentration of the compound in the body, over thecourse of time.

In the discovery and preclinical phase of pharmaceutical drugdevelopment, animal models such as the mouse, rat, monkey, dog, or pig,may be used to perform this characterisation. Any of these models can beused to test the pharmacokinetic properties of the derivatives of theinvention.

The estimate of terminal half-life and/or clearance is relevant forevaluation of dosing regimens and an important parameter in drugdevelopment, in the evaluation of new drug compounds.

Pharmacokinetics Profile—Half Life In Vivo in Minipigs

According to the third functional aspect, the derivatives of theinvention have improved pharmacokinetic properties.

In a particular embodiment, the pharmacokinetic properties may bedetermined as terminal half-life (T_(1/2)) in vivo in minipigs afteri.v. administration, e.g. as described in Example 33 herein.

In one aspect of the invention, the terminal half-life in minipigs is atleast 10 hours. In one aspect of the invention, the terminal half-lifein minipigs is at least 20 hours. In yet another aspect of theinvention, the terminal half-life in minipigs is at least 40 hours.

Production of PYY Compounds

The production of peptides like the PYY compounds of the presentinvention is well known in the art.

The PYY moiety of the derivatives of the invention may for instance beproduced by classical peptide synthesis, e.g., solid phase peptidesynthesis using t-Boc or Fmoc chemistry or other well establishedtechniques, see, e.g., Greene and Wuts, “Protective Groups in OrganicSynthesis”, John Wiley & Sons, 1999, Florencio Zaragoza Dörwald,“Organic Synthesis on solid Phase”, Wiley-VCH Verlag GmbH, 2000, and“Fmoc Solid Phase Peptide Synthesis”, Edited by W. C. Chan and P. D.White, Oxford University Press, 2000.

Also, or alternatively, they may be produced by recombinant methods,viz. by culturing a host cell containing a DNA sequence encoding theanalogue and capable of expressing the peptide in a suitable nutrientmedium under conditions permitting the expression of the peptide.Non-limiting examples of host cells suitable for expression of thesepeptides are: Escherichia coli, Saccharomyces cerevisiae, as well asmammalian BHK or CHO cell lines.

The PYY compounds of the invention which include non-natural amino acidsand/or covalently attached substituents may e.g. be produced asdescribed in the experimental part.

Specific examples of methods of preparing a number of the PYY compoundsof the invention are included in the experimental part.

Protein Purification

The PYY compounds of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic, andreverse-phase high performance liquid chromatography (RP-HPLC)),electrophoretic procedures, or extraction (see, e.g., ProteinPurification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, NewYork, 1989).

Mode of Administration

The term “treatment” is meant to include both the prevention andminimization of the referenced disease, disorder, or condition (i.e.,“treatment” refers to both prophylactic and therapeutic administrationof the PYY compounds of the invention or composition comprising the PYYcompounds of the invention) unless otherwise indicated or clearlycontradicted by context.

The route of administration may be any route which effectivelytransports a compound of this invention to the desired or appropriateplace in the body, such as parenterally, for example, subcutaneously,intramuscularly or intraveneously. Alternatively, a compound of thisinvention can be administered orally, pulmonary, rectally,transdermally, buccally, sublingually, or nasally.

Pharmaceutical Compositions

Injectable compositions comprising PYY compounds of the presentinvention can be prepared using the conventional techniques of thepharmaceutical industry which involve dissolving and mixing theingredients as appropriate to give the desired end product. Thus,according to one procedure, a PYY compound of this invention isdissolved in a suitable buffer at a suitable pH so precipitation isminimised or avoided. The injectable composition is made sterile, forexample, by sterile filtration.

A composition may be a stabilised formulation. The term “stabilisedformulation” refers to a formulation with increased physical and/orchemical stability, preferably both. In general, a formulation must bestable during use and storage (in compliance with recommended use andstorage conditions) until the expiration date is reached.

The term “physical stability” refers to the tendency of the polypeptideto form biologically inactive and/or insoluble aggregates as a result ofexposure to thermo-mechanical stress, and/or interaction withdestabilising interfaces and surfaces (such as hydrophobic surfaces).The physical stability of an aqueous polypeptide formulation may beevaluated by means of visual inspection, and/or by turbiditymeasurements after exposure to mechanical/physical stress (e.g.agitation) at different temperatures for various time periods.Alternatively, the physical stability may be evaluated using aspectroscopic agent or probe of the conformational status of thepolypeptide such as e.g. Thioflavin T or “hydrophobic patch” probes.

The term “chemical stability” refers to chemical (in particularcovalent) changes in the polypeptide structure leading to formation ofchemical degradation products potentially having a reduced biologicalpotency, and/or increased immunogenic effect as compared to the intactpolypeptide. The chemical stability can be evaluated by measuring theamount of chemical degradation products at various time-points afterexposure to different environmental conditions, e.g. by SEC-HPLC, and/orRP-HPLC.

In one aspect, the invention provides PYY compounds with improvedphysical stability. In one aspect, the invention provides PYY compoundswith improved chemical stability.

Combination Treatment

The treatment with a PYY compound according to the present invention mayalso be combined with one or more additional pharmacologically activesubstances, e.g. selected from antidiabetic agents, antiobesity agents,appetite regulating agents, antihypertensive agents, agents for thetreatment and/or prevention of complications resulting from orassociated with diabetes and agents for the treatment and/or preventionof complications and disorders resulting from or associated withobesity.

Examples of these pharmacologically active substances are: GLP-1receptor agonists, insulin, DPP-IV (dipeptidyl peptidase-IV) inhibitors,amylin agonists and leptin receptor agonists.

Pharmaceutical Indications

The present invention also relates to a PYY compound of the inventionfor use as a medicament.

In particular aspects of the invention, the PYY compounds of theinvention may be used for the following medical treatments:

-   -   (i) prevention and/or treatment of all forms of diabetes, such        as hyperglycemia, type 2 diabetes, impaired glucose tolerance,        type 1 diabetes, non-insulin dependent diabetes, MODY (maturity        onset diabetes of the young), gestational diabetes, and/or for        reduction of HbA1C;    -   (ii) delaying or preventing diabetic disease progression, such        as progression in type 2 diabetes, delaying the progression of        impaired glucose tolerance (IGT) to insulin requiring type 2        diabetes, delaying or preventing insulin resistance, and/or        delaying the progression of non-insulin requiring type 2        diabetes to insulin requiring type 2 diabetes;    -   (iii) improving β-cell function, such as decreasing β-cell        apoptosis, increasing β-cell function and/or β-cell mass, and/or        for restoring glucose sensitivity to β-cells;    -   (iv) prevention and/or treatment of eating disorders, such as        obesity, e.g. by decreasing food intake, reducing body weight,        suppressing appetite, inducing satiety; treating or preventing        binge eating disorder, bulimia nervosa, and/or obesity induced        by administration of an antipsychotic or a steroid; reduction of        gastric motility; delaying gastric emptying; increasing physical        mobility; and/or prevention and/or treatment of comorbidities to        obesity, such as osteoarthritis and/or urine incontinence;    -   (v) prevention and/or treatment of diabetic complications, such        as angiopathy; neuropathy, including peripheral neuropathy;        nephropathy; and/or retinopathy;    -   (vi) improving lipid parameters, such as prevention and/or        treatment of dyslipidemia, lowering total serum lipids;        increasing HDL; lowering small, dense LDL; lowering VLDL;        lowering triglycerides; lowering cholesterol; lowering plasma        levels of lipoprotein a (Lp(a)) in a human; inhibiting        generation of apolipoprotein a (apo(a)) in vitro and/or in vivo;    -   (vii) prevention and/or treatment of cardiovascular diseases;        and/or    -   (viii) prevention and/or treatment of sleep apnoea.    -   The following indications are particularly preferred: Type 2        diabetes, and/or obesity.

In one aspect, a method is disclosed herein for altering energymetabolism in a subject. The method includes administering atherapeutically effective amount of a PYY compound of the invention tothe subject, thereby altering energy expenditure. Energy is burned inall physiological processes. The body can alter the rate of energyexpenditure directly, by modulating the efficiency of those processes,or changing the number and nature of processes that are occurring. Forexample, during digestion the body expends energy moving food throughthe bowel, and digesting food, and within cells, the efficiency ofcellular metabolism can be altered to produce more or less heat.

In one aspect a method is disclosed herein for any and all manipulationsof the accurate circuitry described in this application, which alterfood intake co-ordinately and reciprocally alter energy expenditure.Energy expenditure is a result of cellular metabolism, proteinsynthesis, metabolic rate, and calorie utilization. Thus, in thisembodiment, peripheral administration results in increased energyexpenditure, and decreased efficiency of calorie utilization. In oneaspect, a therapeutically effective amount of a PYY compound accordingto the invention is administered to a subject, thereby increasing energyexpenditure.

While “obesity” is generally defined as a body mass index over 30, forpurposes of this disclosure, any subject, including those with a bodymass index of less than 30, who needs or wishes to reduce body weight isincluded in the scope of “obese.” Without intending to be limited bytheory, it is believed that the effects of peripherally administered PYYcompounds of the present invention in the reduction of food intake, inthe delay of gastric emptying, in the reduction of nutrientavailability, and in the causation of weight loss are determined byinteractions with one or more unique receptor classes in, or similar to,those in the PP family. More particularly, it appears that a receptor orreceptors similar to the PYY-preferring (or Y2) receptors are involved.

Particular Embodiments

The invention is further described by the following non-limitingembodiments of the invention:

1. A PYY compound comprising L-beta-homoarginine at a positioncorresponding to position 35 of hPYY(1-36) (SEQ ID NO:1), and apharmaceutically acceptable salt thereof.

2. A PYY compound according to embodiment 1, wherein the PYY compoundhas a maximum of 10 amino acid modifications as compared to hPYY(3-36).

3. A PYY compound according to any one of the preceding embodiments,wherein the PYY compound has a maximum of 8 amino acid modifications ascompared to hPYY(3-36).

4. A PYY compound according to any one of the preceding embodiments,wherein the PYY compound has a minimum of 4 amino acid modifications ascompared to hPYY(3-36).

5. A PYY compound according to any one of the preceding embodiments,wherein the PYY compound has a minimum of 6 amino acid modifications ascompared to hPYY(3-36).

6. A PYY compound according to any one of the preceding embodiments,wherein the PYY compound has a minimum of 8 amino acid modifications ascompared to hPYY(3-36).

7. A PYY compound according to any one of the preceding embodiments,wherein the PYY compound has in the range of 4 to 10 amino acidmodifications as compared to hPYY(3-36).

8. A PYY compound according to any one of the preceding embodiments,wherein the PYY compound has in the range of 6 to 8 amino acidmodifications as compared to hPYY(3-36).

9. A PYY compound according to any one of the preceding embodiments,wherein the PYY compound has 6 amino acid modifications as compared tohPYY(3-36).

10. A PYY compound according to any one of the preceding embodiments,wherein the PYY compound has 8 amino acid modifications as compared tohPYY(3-36).

11. A PYY compound according to any one of the preceding embodiments,wherein the PYY compound exhibit at least 70% sequence identity tohPYY(3-36).

12. A PYY compound according to any one of the preceding embodiments,wherein the PYY compound exhibit at least 75% sequence identity tohPYY(3-36).

13. A PYY compound according to any one of the preceding embodiments,wherein the PYY compound exhibit at least 80% sequence identity tohPYY(3-36).

14. A PYY compound according to any one of the preceding embodiments,wherein the positions corresponding to position 1 and 2 of hPYY(1-36)(SEQ ID NO: 1) are absent.

15. A PYY compound according to any one of the preceding embodiments,wherein the positions corresponding to position 1-3 of hPYY(1-36) (SEQID NO: 1) are absent.

16. A PYY compound according to any one of the preceding embodiments,further comprising arginine at a position corresponding to position 4 ofhPYY(1-36) (SEQ ID NO: 1).

17. A PYY compound according to any one of the preceding embodiments,further comprising glutamine at a position corresponding to position 18of hPYY(1-36) (SEQ ID NO: 1).

18. A PYY compound according to any one of the preceding embodiments,further comprising Aib at a position corresponding to position 22 ofhPYY(1-36) (SEQ ID NO: 1).

19. A PYY compound according to any one of the preceding embodiments,further comprising alanine at a position corresponding to position 24 ofhPYY(1-36) (SEQ ID NO: 1).

20. A PYY compound according to any one of the preceding embodiments,further comprising Aib at a position corresponding to position 24 ofhPYY(1-36) (SEQ ID NO: 1).

21. A PYY compound according to any one of the preceding embodiments,further comprising tryptophane at a position corresponding to position30 of hPYY(1-36) (SEQ ID NO: 1).

22. A PYY compound according to any one of the preceding embodiments,wherein the positions corresponding to positions 1-3 of hPYY(1-36) (SEQID NO: 1) are absent, and wherein the PYY compound further comprises anN-terminal substituent, wherein the N-terminal substituent is an alkoxygroup comprising up to 12 carbon atoms.

23. A PYY compound according to embodiment 22, wherein the N-terminalsubstituent is an alkoxy group comprising up to 10 carbon atoms.

24. A PYY compound according to embodiment 22, wherein the N-terminalsubstituent is an alkoxy group comprising up to 8 carbon atoms.

25. A PYY compound according to embodiment 22, wherein the N-terminalsubstituent is an alkoxy group comprising up to 6 carbon atoms.

26. A PYY compound according to embodiment 22, wherein the N-terminalsubstituent is an alkoxy group comprising 6 carbon atoms.

28. A PYY compound according to embodiment 27, wherein the N-terminalsubstituent is 3-methylbutanoyl.

28. A PYY compound according to any one of the preceding embodiments,further comprising a Lysine at a position corresponding to position 7 ofhPYY(1-36) (SEQ ID NO: 1) and a modifying group attached to the epsilonamino group of the Lysine residue in position 7, wherein said modifyinggroup is defined by A-B-C-, wherein A- comprises a carboxylic acid or asulfonic acid.

29. A PYY compound according to embodiment 28, wherein A- is selectedfrom

wherein a is an integer from 12 to 19, and b is an integer from 10 to16, and wherein * denotes the attachment point to -B-.

30. A PYY compound according to embodiment 29, wherein a is 15, and c is13.

31. A PYY compound according to embodiment 28, wherein A- is

wherein a is an integer from 12 to 19, and wherein * denotes theattachment point to —B—.

32. A PYY compound according to embodiment 31, wherein a is 15.

33. A PYY compound according to embodiment 29, wherein A- is

wherein b is an integer from 10 to 16, and wherein * denotes theattachment point to -B-.

34. A PYY compound according to embodiment 33, wherein b is 13.

35. A PYY compound according to any one of embodiments 28-34, wherein B-is selected from

wherein c is 1 or 2; and d is 1 or 2; and wherein *** denotes theattachment point to A-, and ** denotes the attachment point to -C-.

36. A PYY compound according to embodiment 35, wherein B- is

wherein c is 1 or 2; and wherein *** denotes the attachment point to A-,and ** denotes the attachment point to -C-.

37. A PYY compound according to any one of embodiments 28-36, wherein-C- is

wherein e is an integer in the range of 1-5, f is an integer in therange of 1-5, g is an integer in the range of 2 to 6, and wherein ****denotes the attachment point to -B-, and ***** denotes the attachmentpoint to the epsilon amino group of the Lysine residue in the positioncorresponding to position 7 of hPYY(1-36).

38. A PYY compound according to embodiment 37, wherein e and f are each1.

39. A PYY compound according to any one of embodiments 37-38, wherein gis selected from 2, 4 or 6.

40. A PYY compound according to embodiment 39, wherein g is 2.

41. A PYY compound according to embodiment 39, wherein g is 4.

42. A PYY compound according to embodiment 39, wherein g is 6.

43. A PYY compound according to any one of the preceding embodiments,wherein the PYY compound is not a salt.

44. A PYY compound according to any one of the preceding embodimentsselected from the following:

45. A PYY compound according to any one of the preceding embodimentswhich is a human Y2 receptor agonist.

46. A PYY compound according to any one of the preceding embodimentswhich is a full human Y2 receptor agonist.

47. A PYY compound according to any one of the preceding embodimentswhich is a selective human Y2 receptor agonist.

48. A PYY compound according to any one of the preceding embodimentswhich is a selective full human Y2 receptor agonist.

49. A PYY compound according to any one of the preceding embodimentswhich is capable of activating the human Y2 receptor.

50. A PYY compound according to any one of the preceding embodimentswhich is capable of activating the human Y2 receptor in an assay withwhole cells expressing the human Y2 receptor.

51. A PYY compound according to any one of the preceding embodimentswhich is capable of activating the human Y2 receptor in the Actonefunctional potency assay of example 31.

52. A PYY compound according to any one of the preceding embodimentswhich is capable of binding to the human Y2 receptor.

53. A PYY compound according to any one of the preceding embodimentswhich is capable of binding to the human Y2 receptor, wherein thebinding to the human Y2 receptor is measured in a competitive bindingassay, such as the assay of example 32.

54. A PYY compound according to any one of the preceding embodimentswhich has improved pharmacokinetic properties.

55. A PYY compound according to any one of the preceding embodimentswhich has an increased half-life and/or a decreased clearance.

56. A PYY compound according to any one of the preceding embodimentswhich has the effect in vivo of decreasing the blood glucose determinedin a single-dose study in a db/db mouse model.

57. A PYY compound according to any one of the preceding embodimentswhich has the effect in vivo of decreasing food intake determined in asingle-dose study in a db/db mouse model.

58. A PYY compound according to any one of embodiments 1-44, for use asa medicament.

59. A PYY compound according to any one of embodiments 1-44, for use inthe treatment and/or prevention of all forms of diabetes and relateddiseases, such as eating disorders, diabetic complications,cardiovascular diseases and/or sleep apnoea; and/or for improving lipidparameters, improving β-cell function, and/or for delaying or preventingdiabetic disease progression.

60. The use of a PYY compound according to any one of embodiments 1-44,for use in the manufacture of a medicament for the treatment and/orprevention of all forms of diabetes and related diseases, such as eatingdisorders, diabetic complications, cardiovascular diseases and/or sleepapnoea; and/or for improving lipid parameters, improving β-cellfunction, and/or for delaying or preventing diabetic diseaseprogression.

61. A method of treatment and/or prevention of all forms of diabetes andrelated diseases, such as eating disorders, diabetic complications,cardiovascular diseases and/or sleep apnoea; and/or for improving lipidparameters, improving β-cell function, and/or for delaying or preventingdiabetic disease progression by administering a pharmaceutically activeamount of a PYY compound according to any one of embodiments 1-44.

EXAMPLES

This experimental part starts with a list of abbreviations, and isfollowed by a section including general methods for synthesising andcharacterising compounds of the invention. Then follows a number ofexamples which relate to the preparation of specific PYY compounds, andat the end a number of examples have been included relating to theactivity and properties of these compounds (section headedpharmacological methods).

The examples serve to illustrate the invention.

LIST OF ABBREVIATIONS

Aib: α-aminoisobutanoic acid

r.t: Room temperature

ACN: acetonitrile

DIPEA: diisopropylethylamine

H₂O: water

CH₃CN: acetonitrile

DMF: N,N-dimethylformamide

Fmoc: 9H-fluoren-9-ylmethoxycarbonyl

Boc: tert butyloxycarbonyl

OtBu: tert butyl ester

tBu: tert butyl

Trt: triphenylmethyl

Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl

PyBOP: benzotriazol-1-yl-oxytripyrrolidinophosphoniumhexafluorophosphate

HFIP: Hexafluoroisopropanol

Mtt: 4-methyltrityl

DCM: dichloromethane

TIPS: triisopropylsilane

TFA: trifluoroacetic acid

Et2O: diethyl ether

NMF: 1-Methyl-formamide

NMP: 1-Methyl-pyrrolidin-2-one

DIPEA: Diisopropylethylamine

HOAc: acetic acid

HOAt: 1-Hydroxy-7-azabenzotriazole

HOBt: 1-Hydroxybenzotriazole

DIC: Diisopropylcarbodiimide

Min: minutes

MW: Molecular weight

HMWP: High molecular weight proteins

beta-homoArg: L-beta-homoarginine

Materials and Methods

General Methods of Preparation

This section relates to methods for solid phase synthesis of peptidebackbone and synthesis of side chain attached to backbone (SPPS methods,including methods for the coupling of amino acids, the de-protection ofFmoc-amino acids, methods for cleaving the peptide from the resin, andfor its purification).

1. Synthesis of Resin Bound Protected Peptide Backbone

Procedure for the Automatic Step-Wise Assembly of Peptide Backbone.

The protected peptidyl resin was synthesized according to the Fmocstrategy on a solid phase peptide synthesiser Prelude (ProteinTechnologies, Tucson, USA) either 0.25 mmol scale or 0.4 mmol scaleusing the manufacturer supplied machine protocols. The Fmoc-protectedamino acid derivatives used were the standard recommended: Fmoc-Ala-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH,Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH,Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Mtt)-OH,Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH,Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, or, Fmoc-Val-OHetc. supplied from e.g., Bachem, Iris Biotech, Protein Technologies orNovabiochem. If nothing else is specified the natural L-form of theamino acids are used. Coupling was done by the use of DIC(dicyclohexylcarbodiimide) and Ozyma Pure (ethyl2-cyano-2-(hydroxyimino)-acetate, Merck, Novabiochem, Switzerland)mediated couplings in NMP (N-methyl pyrrolidone). The coupling of theFmoc-amino acid was done as described above using 4-8 time excess ofamino acid relative to resin substitution (4-8 eq). Coupling time rangedfrom 1 hour up to 4 hours. The Fmoc-Arg(pbf)-OH was coupled using adouble coupling procedure (1 hour+1 hour). The resin used for thesynthesis of the peptide amides can be Tentagel RAM (Rapp Polymere,Germany), Rink amid ChemMatrix resin (Matrix Innovation, Canada)Rink-Amide resin (Merck/Novabiochem). The protected amino acidderivatives used were standard Fmoc-amino acids (supplied from e.g.Protein Technologies, or Novabiochem. The epsilon amino group of lysineto be derivatised was protected with Mtt. The N-terminal amino acid orbuilding was coupled as a Boc-protected amino acid, e.g., Boc-Ile.Alternatively isovaleric acid was coupled according to the abovedescribed coupling procedure for the Fmoc-amino acids. The step-wisesolid phase assembly on the Prelude was done using the followingsteps: 1) deprotection (removal of Fmoc) by the use of 25% piperidine inNMP for 2×4 min., step 2) Wash (removal of piperidine) with NMP and DCM,step 3) Coupling of Fmoc-amino acid (0.3M Fmoc-amino acid in 0.3M OxymaPure in NMP) 4-8 eq excess for 1-4 hours coupling initiated by adding1/10 volume of 3M DIC in NMP and 1/10 volume collidine in NMP. Mixingwas done by occasional bubbling with nitrogen, step 4) Wash (removal ofexcess amino acid and reagents by the use of NMP and DCM). Last stepincluded washing with DCM which made the resin ready for attachment ofalbumin binding moiety on lysine side chain.

2. Attachment of Modifying Groups to Resin Bound Protected PeptideBackbone

Procedure for manual removal of Mtt-protection (lysine(Mtt):

Before synthesis of the modifying group, the Mtt group on the site ofattachment (lysine) must be removed. The resin was placed in a syringeor reaction flask and treated with 75% hexafluroisopropanol (HFIP)+25%DCM for 2×30 min to remove the Mtt group. The resin was then washed withDCM and NMP as described above and neutralized with 5% DIPEA(neutralisation step) in NMP or 25% piperidine in NMP followed by NMPwashing before coupling the albumin moiety. Alternatively, theneutralisation step was omitted.

Procedure for Prelude Removal of Mtt-Protection (Lysine(Mtt)):

On the Prelude the resin was treated with 75% hexafluoroisopropanol(HFIP)+25% DCM for 2×2 min followed 2×30 min to remove the Mtt group onthe lysine. The resin was then washed with DCM and NMP followed by aneutralisation step using 25% piperidine in NMP by 4 min, and was thenready for the synthesis of the modifying group.

Procedure for Manual Synthesis of Modifying Groups onto a LysineResidue:

The building blocks Fmoc-8-amino-3,6-dioxaoctanoic acid, Fmoc-Glu-OtBu,and eicosanedioic acid mono-tert-butyl ester (CAS No. 843666-40-0) werecoupled using DIC and Oxyma Pure in 4-8 eq relative to resinsubstitution. The coupling time was 2-16 hours usually followed by acapping step using 1 M acetic anhydride for 15-60 min. The Fmoc-groupwas removed by 25% piperidine in NMP for 10-30 min. followed by washing.The 16-sulfonic hexadecanoic acid was solubilised in NMP orN-methylformamid (NMF) at 60 degree Celsius or above and activated byPyBOP 1 eq relative to the sulfonic hexadecanoic acid and 2 eq ofdiisopropylethylamine (DIPEA) relative to sulfonic hexadecanoic acid wasalso added. The peptidyl resin was washed with hot NMP or NMF just priorto the addition of activated sulfonic hexadecanoic acid. An excess of3-4 of the sulfonic building block was used and coupling allowed toproceed >16 hours.

Procedure for Automated Synthesis of Modifying Groups onto a LysineResidue:

For the synthesis of the modifying groups the following building blockswere used: Fmoc-8-amino-3,6-dioxaoctanoic acid, Fmoc-Glu-OtBu, andeicosanedioic acid mono-tert-butyl ester (CAS No. 843666-40-0).Modifying groups were coupled using DIC and Oxyma Pure in 4-8 eqrelative to resin substitution. The coupling time was 2-16 hours usuallyfollowed by a capping step using 1 M acetic anhydride for 20 min. TheFmoc-group was removed by 25% piperidine in NMP for 2×4 min. followed bywashing as described in the SPPS of the peptide backbone. All othersynthesis steps were also the same as described above with the backbonesynthesis. The coupling of 16-sulfonic hexadecanoic acid was done by themanual procedure as described above using pyBOP as coupling reagent.

3. Cleavage of Resin Bound Peptide with or without Attached ModifyingGroups and Purification

Prior to TFA deprotection the peptidyl resin was washed with DCM ordiethyl ether and dried. The peptide and side chain protection groupswere removed by addition of 20-40 ml (0.25 mmol scale) 30-60 (0.4 mmolscale) ml 92% TFA, 5% TIPS and 3% H₂O for 2-4 hours. Then TFA wasfiltered and in some cases concentrated by a stream of argon and diethylether was added to precipitate the peptide. The peptide was washedthree-five times with diethyl ether and dried.

General Methods of Detection and Characterisation

This section relates to methods for detection and characterisation ofthe resulting peptides, including LCMS, MALDI and UPLC methods.

1. LC-MS method (LCMS1)

Agilent Technologies LC/MSD TOF (G1969A) mass spectrometer was used toidentify the molecular weight of the peptide after elution from anAgilent 1200 series HPLC system. The de-convolution of the mass data wascalculated using the Agilents software.

Eluents:

Buffer A: 0.1% TFA in water

Buffer B: 0.1% TFA in CH₃CN

LC-MS Waters Acquity (LCMS2)

LC-system: Waters Acquity UPLC

Column: Waters Acquity UPLC BEH, C-18, 1.7 μm, 2.1 mm×50 mm

Detector: Waters (Micromass) LCT Premier XE

Linear gradient: 5% to 95% B

Gradient run-time: 4.0 minutes

Total run-time: 7.0 minutes

Flow rate: 0.4 ml/min

Column temperature: 40° C.

Solvent A: 99.90% MQ-water, 0.1% formic acid

Solvent B: 99.90% acetonitrile, 0.1% formic acid

2. UPLC Methods

Method UPLC26v01

Buffer A: 0.05% TFA

Buffer B: CH3CN+0.05% TFA

Flow: 0.45 ml/min

Gradient: 5-60% Buffer B (0.5-4 min.)

Column: Acquity UPLC BEH C18 1.7 um, 2.1×50 mm

Column temperature: 40° C.

Method UPLC29v01

Buffer A: 0.05% TFA

Buffer B CH3CN+0.05% TFA

Flow: 0.45 ml/min

Gradient: 15-35% Buffer B (0.5-4 min.)

Column: Acquity UPLC BEH C18 1.7 um, 2.1×50 mm

Column temperature: 40° C.

Method UPLC30v01

Buffer A: 0.05% TFA

Buffer B: CH3CN+0.05% TFA

Flow: 0.45 ml/min

Gradient: 20-40% Buffer B (0.5-4 min.)

Column: Acquity UPLC BEH C18 1.7 um, 2.1×50 mm

Column temperature: 40° C.

Method UPLC31v01

Buffer A: 0.05% TFA

Buffer B: CH3CN+0.05% TFA

Flow: 0.45 ml/min

Gradient: 25-45% Buffer B (0.5-4 min.)

Column: Acquity UPLC BEH C18 1.7 um, 2.1×50 mm

Column temperature: 40° C.

Method UPLC02v01

System: Waters Acquity UPLC system

Buffer A: 0.05% TFA in H₂O

Buffer B: CH3CN+0.05% TFA

Flow: 0.40 ml/min

Gradient: 5-95% Buffer B (16 min.)

Column: Acquity UPLC BEH C18 1.7 um, 2.1×150 mm

Column temperature: 40° C.

Method UPLC07v01

System: Waters Acquity UPLC system

Buffer A: 0.09 M di-Ammonium Hydrogen Phosphate (aq) and 10%Acetonitrile, pH 3.6

Buffer B: 20% Isopropanole, 20% Water and 60% Acetonitrile

Flow: 0.50 ml/min

Gradient: 35-65% Buffer B (2-17 min.)

Column: Phenomenex Kinetex C18, 1.7 um, 2.1 mm×150 mm column

Column temperature: 60° C.

Method UPLC16v01

System: Waters Acquity UPLC system

Buffer A: 0.2 M Sodium Sulfate, 0.02 M di-Sodium Hydrogen Phosphate,0.02 M Sodium

di-Hydrogen Phosphate, 90% Water and 10% Acetonitrile, pH 7.2

Buffer B: 70% Acetonitrile, 30% Water

Flow: 0.40 ml/min

Gradient: 20-50% Buffer B (3-20 min.)

Column: ACQUITY UPLC BEH Shield RP18, 1.7 um, 2.1 mm×150 mm column

Column temperature: 60° C.

Method UPLC17v01

System: Waters Acquity UPLC system

Buffer A: 0.2 M Sodium Sulfate, 0.02 M di-Sodium Hydrogen Phosphate,0.02 M Sodium

di-Hydrogen Phosphate, 90% Water and 10% Acetonitrile, pH 7.2

Buffer B: 70% Acetonitrile, 30% Water

Flow: 0.40 ml/min

Step gradient: 10-20% B over 3 minutes, then 20-80% B over 17 minutes,then 80-90%

B over 1 minute

Column: ACQUITY UPLC BEH Shield RP18, 1.7 um, 2.1 mm×150 mm column

Column temperature: 60° C.

Method UPLC-AP-01

Buffer A: 0.1% TFA in H₂O

Buffer B: CH3CN+0.1% TFA

Flow: 0.40 ml/min

Gradient: 5-95% Buffer B (16 min.)

Column; Acquity UPLC BEH130; 150×2.1; 1.7 um

Column temperature: 40° C.

Method UPLC-AP-02

Buffer A: 20 mM Na2HPO4, 20 mM NaH2PO4, 200 mM Na2SO4 in 90% water/10%acetonitrile, pH 7.20

Buffer B: 70% acetonitrile/ 30% water

Flow: 0.40 ml/min

Gradient: 10-20% Buffer B (0-3 min.); 20-50% buffer B (3-20 min); 50-80%(20-21 min)

Column; Acquity UPLC BEH Shield, RP18 1.7 um, 2.1×150 mm

Column temperature: 40° C.

3. MALDI-MS Method

Molecular weights of the peptides were determined using matrix-assistedlaser desorption time of flight mass spectroscopy (MALDI-MS), recordedon a Microflex (Bruker). A matrix of alpha-cyano-4-hydroxy cinnamic acidwas used. The molecular weight of the product was calculated based onthe result of MALDI-MS analysis using the software supplied from themanufacturer.

Synthesis of Intermediates Synthesis of 16-sulfo-hexadecanoic acid

16-Hexadecanolide (997 g, 3.92 mol) was dissolved in methanol (15.1 L)and toluene-4-sulfonic acid monohydrate (90.0 g, 0.473 mol) was added.Reaction mixture was heated in 50 L reactor at 55° C. for 16 hours.After cooling down sodium hydrogen carbonate (56.0 g, 0.67 mol) wasadded and the reaction mixture was stirred for 15 min. Solvent wasevaporated on Heidolph 20 L rotary evaporator. Ethyl acetate (12 L) wasadded and the mixture was extracted with 5% solution of sodium hydrogencarbonate (10 L). Organic layer was separated; emulsion layer wasextracted with ethyl acetate (3×3 L), white insoluble muddy material wasseparated and ethyl acetate layer was washed again with 5% solution ofsodium hydrogen carbonate (5 L).Organic layers were combined and washedwith saturated solution of sodium hydrogen carbonate (5 L) and brine (10L). Solvent was evaporated on Heidolph 20 L rotary evaporator. Crudeproduct was crystallized from hexanes (8 L). Hot solution in hexanes wasdecanted and then let to crystallize in ice bath. The material wasfiltered on large frit and washed with cold hexanes (2 L). Pure materialwas dried in vacuo.

Yield: 1062.2 g (95%). R_(F) (SiO₂, dichloromethane/methanol 95:5):0.65.

¹H NMR spectrum (300 MHz, CDCl₃, δ_(H)): 3.67 (s, 3H); 3.67-3.60 (m,2H); 2.30 (t, J=7.5 Hz, 2H); 1.67-1.53 (m, 4H); 1.25 (s, 22H).

The above ester (957 g, 3.34 mol) was dissolved in dichloromethane (7 L)on Heidolph 20 L rotary evaporator. Triethylamine (695 mL, 4.98 mol) wasadded, reaction mixture was cooled to 0° C. (by putting ice intoevaporator bath) and methanesulfonyl chloride (325 mL, 4.19 mol) indichloromethane (200 mL) was added slowly during 10 minutes by externaltubing using small vacuum. Then the reaction mixture was heated to 35°C. for 1 hour. NMR analysis showed complete conversion. Water was added(690 mL) and solvents were evaporated. Ethyl acetate (8 L) was added andthe mixture was washed with 1 M hydrochloric acid (4 L) and 5% solutionof sodium carbonate (4 L). Since sodium carbonate extraction formed anemulsion this layer was extracted with ethyl acetate (4 L) and added tomain portion. Combined ethyl acetate layer was washed with brine (4 L),dried over anhydrous sodium sulfate and filtered. Solvent was evaporatedgiving 16-methanesulfonyloxy-hexadecanoic acid methyl ester as whitesolid.

Yield: 1225.4 g (100%).

¹H NMR spectrum (300 MHz, CDCl₃, δ_(H)): 4.22 (t, J=6.6 Hz, 2H); 3.66(s, 3H); 3.00 (s, 3 H); 2.30 (t, J=7.5 Hz, 2H) 1.82-1.67 (m, 2H);1.68-1.54 (m, 2H); 1.36-1.17 (m, 22 H).

The above mesylate (1.23 kg, 3.34 mol) was dissolved in acetone (8 L)and lithium bromide (585 g, 6.73 mol) was added and the reaction mixturewas heated on Heidolph 20 L rotary evaporator at 50° C. for 12 hours.After cooling down solvent was evaporated, ethyl acetate (10 L) wasadded and the mixture was washed with 5% solution of sodiumhydrogencarbonate (3×15 L) and brine (8 L). Solvent was evaporated todryness to yield 16-bromo-hexadecanoic acid methyl ester as pale yellowoil which started to crystallize.

Yield: 1219 g (105%); contains acetone and product of acetonealdolization.

R_(F) (SiO₂, hexanes/ethyl acetate 9:1): 0.90.

¹H NMR spectrum (300 MHz, CDCl₃, δ_(H)): 3.65 (s, 3H); 3.42 (t, J=6.9Hz, 2H); 2.32 (t, J=7.5 Hz, 2H); 1.92-1.77 (m, 2H) 1.69-1.53 (m, 2H);1.50-1.35 (m, 2H); 1.25 (bs, 10H).

Solutions of sodium sulfite (327 g, 2.60 mol) in water (1.26 L) and16-bromo-hexadecanoic acid methyl ester (728 g, 2.00 mol, 96% purity) in1-propanol (945 mL) and methanol (420 mL) were heated to reflux in 6 Lreactor equipped with mechanical stirrer for 48 hours. The reactionmixture was cooled to 27° C. and diluted with tetrahydrofuran (2 L).Reaction mixture was filtered and solid material was washed withtetrahydrofuran (3×700 mL). Filtrate was cooled to 0° C. and anotherportion of material was precipitated. This precipitate was filtered andwashed with tetrahydrofuran (2×200 mL). Solids were combined and mixedwith water (8.4 L) in 20 L pot. Solution of sodium hydroxide (120 g,3.00 mol) was added. The mixture was heated to boiling for about 5hours. Solution of sulfuric acid (430 mL, 8.00 mol) in water (500 mL)was slowly added into the reaction mixture (sulfur dioxide is formed).Reaction mixture was heated to boiling for 10 min and then let to coolto 15° C. (ice bath). The mixture was filtered on Büchner funnel throughfilter paper Seitz (several layers filter) applying vacuo. Thisprocedure was very slow and took two days. Solid material was severaltimes washed with distilled water until pH of filtrate was between 2 and3. This procedure took about three days. Muddy white material was driedin oven at 80° C. giving desired product.

Yield: 510 g (76%).

¹H NMR spectrum (300 MHz, DMSO-d₆, δ_(H)): 2.45-2.33 (m, 2H); 2.18 (t,J=7.3 Hz, 2H); 1.60-1.40 (m, 4H); 1.24 (s, 22H).

MS-ESI (neg, sample in H₂O/MeCN+NaHCO₃; m/z): 335.5 (M-H)⁻, 357.5(M-2H+Na)⁻, 167.3 (M-2H)²⁻

Synthesis of Compounds of the Invention Example 1

SEQ ID NO:1

hPYY(1-36)

YPIKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY-NH₂

Example 2

SEQ ID NO:2

hPYY(3-36)

IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY-NH₂

Retention time HPLC method UPLC16v01: 3.37 min (91.4%)

Retention time HPLC method UPLC29v01: 10.07 min (85.6%)

MW calculated: 4049.6 g/mol

MALDI MS: 4048.2 g/mol

Example 3

Retention time HPLC method UPLC16v01: 9.35 min (79.4%)

Retention time HPLC method UPLC29v01: 3.3 min (82.3%)

MW calculated: 4049.55 g/mol

MALDI (MALDSI-MS found): 4048.3 g/mol.

Example 4

Retention time HPLC method UPLC16v01: 9.53 min (83.4%)

Retention time HPLC method UPLC29v01: 3.33 min (88.9%)

MW calculated: 4077.61 g/mol

MALDI (MALDSI-MS found): 4076.1 g/mol

Example 5

Retention time HPLC method UPLC16v01: 9.47 min (82.8%)

Retention time HPLC method UPLC29v01: 3.31 min (88%)

MW calculated: 4063.58 g/mol

MALDI (MALDSI-MS found): 4062.8 g/mol

Example 6

Retention time HPLC method UPLC16v01: 9.47 min (92.9%)

Retention time HPLC method UPLC29v01: 3.4 min (97.3%)

Mw calculated: 4063.58 g/mol

MALDI (MALDSI-MS found): 4060.5 g/mol

Example 7

Retention time HPLC method UPLC16v01: 10.25 min (84.4%)

Retention time HPLC method UPLC29v01: 3.39 min (93.9%)

MW calculated: 4136.63 g/mol

LCMS (LCMS1): m/z: 1379.7 ((M/3)+3); 1035.0 ((M/4)+4); 690.4 ((M/6+6)

Example 8

Retention time HPLC method UPLC16v01: 9.70 min (94.1%)

Retention time HPLC method UPLC29v01: 3.37 min (96.5%)

MW calculated: 4077.61 g/mol

MALDI (MALDSI-MS found): 4076.2 g/mol

Example 9

Retention time UPLC-AP-01: 5.92 min (95.1%)

Retention time UPLC-AP-02: 10.23 min (90.6%)

MW calculated: 4150.66 g/mol

LCMS2: ((M/3+3) 1384.3; ((M/4)+4) 1038.5; ((M/5+5) 831.1

Example 10

Retention time UPLC-AP-01: 5.38 min (97.3%)

Retention time UPLC-AP-02: 8.29 min (95.7%)

MW calculated: 4035.53 g/mol

LCMS2: ((M/3+3) 1346.0; ((M/4)+4) 1009.3; ((M/5+5) 808.4

Example 11

Retention time HPLC method UPLC16v01: 11.01 min (94.4%)

Retention time HPLC method UPLC29v01: 3.19 min (100%)

MW calculated: 4049.55 g/mol

LCMS (LCMS1): m/z: 1350.7 ((M/3)+3); 1013.3 ((M/4)+4)

Example 12

Retention time HPLC method UPLC16v01: 8.6 min (81.3%)

Retention time HPLC method UPLC29v01: 3.11 min (98.7%)

MW calculated: 4035.53 g/mol

LCMS (LCMS1): m/z: 1346 ((M/3)+3); 1009.8 ((M/4)+4); 673.5 ((M/6+6)

Example 13

Retention time HPLC method UPLC16v01: 9.05 min (84.4%)

Retention time HPLC method UPLC29v01: 3.19 min (100%)

MW calculated: 4108.58 g/mol

LCMS (LCMS1): m/z: 1370.4 ((M/3)+3); 1028.0 ((M/4)+4)

Example 14

Retention time UPLC-AP-01: 5.61 min (99.3%)

Retention time UPLC-AP-02: 10.0 min (97.2%)

MW calculated: 4077.57 g/mol

LCMS2: ((M/3+3) 1360.1; ((M/4)+4) 1020.5; ((M/5+5) 816.3

Example 15

Retention time UPLC-AP-01: 5.4 min (98%)

Retention time UPLC-AP-02: 9.14 min (95.1%)

MW calculated: 4063.54 g/mol

LCMS2: ((M/3+3) 1355.8; ((M/4)+4) 1016.9; ((M/5+5) 814.4

Example 16

Retention time UPLC-AP-01: 7.34 min (96.7%)

Retention time UPLC-AP-02: 12.37 min (91.4%)

MW calculated: 4836.57 g/mol

LCMS2: ((M/3+3) 1613.0; ((M/4)+4) 1209.3; ((M/5+5) 968.1

Example 17

Retention time HPLC method UPLC16v01: 12.21 min (97.9%)

Retention time HPLC method UPLC30v01: 3.72 min (98.2%)

MW calculated: 4850.59 g/mol

LCMS (LCMS1): m/z: 1617.9 ((M/3)+3); 1213.7 ((M/4)+4); 809.5 ((M/6+6)

Example 18

Retention time HPLC method UPLC16v01: 11.63 min (96.2%)

Retention time HPLC method UPLC30v01: 3.788 min (95.9%)

MW calculated: 5140.91 g/mol

MALDI (MALDI-MS found): 5139.1 g/mol

Example 19

Retention time HPLC method UPLC16v01: 11.45 min (95.1%)

Retention time HPLC method UPLC02v01: 7.26 min (100%)

MW calculated: 5052.76 g/mol

LCMS (LCMS1): m/z: 2527.3 ((M/2)+2); 1685.23 ((M/3)+3); 1264.18((M/4)+4)

Example 20

Retention time HPLC method UPLC16v01: 12.25 min (96.0%)

Retention time HPLC method UPLC02v01: 7.34 min (99.1%)

MW calculated: 4923.65 g/mol

LCMS (LCMS1): m/z: 2462.78 ((M/2)+2); 1642.21 ((M/3)+3); 1231.92((M/4)+4)

Example 21

Retention time HPLC method UPLC16v01: 12.37 min (91.5%)

Retention time HPLC method UPLC30v01: 3.88 min (93%)

MW calculated: 4864.62 g/mol

MALDI (MALDI-MS found): 4863.1 g/mol

Example 22

Retention time UPLC-AP-01: 7.31 min (98.8%)

Retention time UPLC-AP-02: 11.8 min (94.1%)

MW calculated: 4864.58 g/mol

LCMS2: ((M/3+3) 1622.7; ((M/4)+4) 1216.8; ((M/5+5) 973.9

Example 23

Retention time UPLC-AP-01: 7.31 min (95.3%)

Retention time UPLC-AP-02: 11.9 min

MW calculated: 4878.61 g/mol

LCMS2: ((M/3+3) 1626.4; ((M/4)+4) 1220.2; ((M/5+5) 976.4

Example 24

Retention time HPLC method UPLC16v01: 11.03 min (98.9%)

Retention time HPLC method UPLC02v01: 7.5 min (100%)

MW calculated: 4950.67 g/mol

LCMS (LCMS1): m/z 1653.23 ((M/3)+3); 1238.6 ((M/4)+4)

Example 25

Retention time HPLC method UPLC16v01: 12.18 min (97.7%)

Retention time HPLC method UPLC02v01: 7.58 min (99.3%)

MW calculated: 4821.55 g/mol

LCMS (LCMS1): m/z 2411.79 ((M/2)+2); 1607.89 ((M/3)+3); 1206.43((M/4)+4)

Example 26

Retention time HPLC method UPLC16v01: 12.56 min (98.5%)

Retention time HPLC method UPLC31v01: 3.37 min (98.8%)

MW calculated: 4894.61 g/mol

LCMS (LCMS1): m/z: 1632.49 ((M/3)+3); 1224.35 ((M/4)+4); 979.67((M/5)+5)

Example 27

Retention time HPLC method UPLC16v01: 12.21 min (83.11%)

Retention time HPLC method UPLC30v01: 3.44 min (90.5%)

MW calculated: 5045.75 g/mol

LCMS (LCMS1): m/z: 1682.85 ((M/3)+3); 1262.37 ((M/4)+4); 1010.09((M/5)+5)

Example 28

Retention time HPLC method UPLC16v01: 11.23 min (88.1%)

Retention time HPLC method UPLC30v01: 3.35 min (91.7%)

MW calculated: 4916.63 g/mol

MALDI (MALDI-MS found): 4914.5 g/mol

Example 29

Retention time HPLC method UPLC16v01: 12.65 min (94.8%)

Retention time HPLC method UPLC32v01: 2.5 min (96.6%)

MW calculated: 4922.62 g/mol

LCMS (LCMS1): m/z: 1641.48 ((M/3)+3); 985.46 ((M/5)+5)

Example 30

Retention time HPLC method UPLC16v01: 11.36 min (83.4%)

Retention time HPLC method UPLC30v01: 3.37 min (93.0%)

MW calculated: 4944.64 g/mol

MALDI (MALDI-MS found): 4944.3 g/mol.

Pharmacological Methods

The utility of PYY peptide derivatives or analogues thereof of thepresent invention as pharmaceutically active agents in the reduction ofweight gain and treatment of obesity in mammals (such as humans), andfor treatment of diabetes may be demonstrated by the activity of theagonists in conventional assays and in the in vitro and in vivo assaysdescribed below.

Such assays also provide a means whereby the activities of the PYYcompounds of this invention can be compared with the activities of knowncompounds.

Example 31 Receptor Potency of PYY Compounds

The purpose of this example is to test the activity, or potency, of thePYY compounds in vitro. The in vitro potency is the measure of theactivation of the human Y1, Y2, Y4 and Y5 receptor subtypes,respectively, in a whole cell assay.

The potencies of the PYY compounds of examples 2-30 were determinedusing the Actone functional potency assay as described below. hPYY(3-36)(Example 2, SEQ ID NO:2) was included as a reference. Compounds ofexamples 3-5 were included as comparative examples.

Actone Functional Potency Assay

The Neuropeptide Y (NPY) receptors are G_(i)-coupled seventrans-membrane receptors that mainly signal through the cAMP dependentpathway by inhibiting adenylate cyclase activity which results in adecrease of cAMP production from ATP. The Actone assay is based on amodified calcium channel that has a selective binding for cAMP,resulting in cellular calcium influx, detected by a calcium responsivedye. In order to measure decreased levels of cAMP, as result of NPYreceptor activation, isoproterenol a β1/β2-adrenoreceptor agonist isadded to activate adenylate cyclase and increase cAMP levels in thecell. Decreased cellular calcium concentrations, reflecting a decreaseof cAMP levels due to NPY receptor activation, is detected as a decreasein fluorescence from the calcium sensitive dye.

HEK-293 cells expressing the cAMP sensitive calcium channel and one ofthe NPYR-Y1, NPYR-Y2, NPYR-Y4 or NPYR-Y5 (CodexBiosolution,Gaithersburg, Md., USA) were seeded into poly lysine coated 384 wellplates at a density of 14.000 cells/well in a volume of 25 μl in DMEMmedium containing 10% Fetal calf serum (FCS), 1%Penicillin-Streptomycin, 250 μg/ml aminoglycoside antibiotic G418 and 1μg/ml aminonucleoside antibiotic puromycin. The cells were incubatedover night at +37° C. in a humidified milieu in 5% CO₂ followed byaddition of 25p1 calcium dye buffer containing: 1 vial Calcium 5 dye(Molecular Devices, Sunnyvale, Calif., USA) solved in 100 ml buffercontaining 20 mM Hepes, 0.1% Ovalbumin, 0.005% Tween 20, 1.5 mMprobenecid, 250 μM PDE-inhibitor4-(3-Butoxy-4-methoxybenzyl)imidazolidin-2-one and 8 mM CaCl₂ and pH wasadjusted to 7.40. Cells were incubated for 1 hour with the calcium dyebuffer and then placed in a FLIPR Tetra System (Molecular Devices) wherethe liquid handling system added analogue (1000-1 nM finalconcentrations) and isoproterenol (0.05 μM final concentration)simultaneously directly followed by fluorescence signal measurement(Ex540/Em590) for 360 seconds with 30 seconds intervals. Allmeasurements were performed in duplicates and EC₅₀ values werecalculated by nonlinear regression analysis of sigmoidal dose responsecurves using the GraphPad Prism v 5.02 (Graph Pad software, La Jolla,Calif., USA). The EC50 values are shown in table 1 below.

TABLE 1 In vitro potency Y2 Y1 Y4 Y5 Example Compound EC50 (nM) EC50(nM) EC50 (nM) EC50 (nM)  2 hPYY(3-36) 0.80 6.6 651 8.4 SEQ ID NO: 2  3SEQ ID NO: 3 3.1 >1000 >1000 3.1 (comparative example)  4 SEQ ID NO: 4798 >1000 >1000 798 (comparative example)  5 SEQ ID NO:5 >1000 >1000 >1000 >1000 (comparative example)  6 SEQ ID NO: 6 5.9831 >1000 816  7 SEQ ID NO: 7 2.4 >1000 >1000 802  8 SEQ ID NO: 8 1.4736 >1000 951  9 SEQ ID NO: 9 3.7 >1000 >1000 854 10 SEQ ID NO: 103.6 >1000 >1000 >1000 11 SEQ ID NO: 11 2.5 >1000 >1000 >1000 12 SEQ IDNO: 12 3.0 >1000 >1000 >1000 13 SEQ ID NO: 13 1.8 >1000 >1000 >1000 14SEQ ID NO: 14 2.5 >1000 >1000 >1000 15 SEQ ID NO: 153.7 >1000 >1000 >1000 16 SEQ ID NO: 16 22 >1000 >1000 907 17 SEQ ID NO:17 7.2 964 >1000 880 18 SEQ ID NO: 18 37 >1000 >1000 >1000 19 SEQ ID NO:19 12 >1000 >1000 >1000 20 SEQ ID NO: 20 3.9 >1000 >1000 >1000 21 SEQ IDNO: 21 9.0 >1000 >1000 >1000 22 SEQ ID NO: 22 28 >1000 >1000 >1000 23SEQ ID NO: 23 32 >1000 >1000 925 24 SEQ ID NO: 24 23 >1000 >1000 >100025 SEQ ID NO: 25 9.1 >1000 >1000 >1000 26 SEQ ID NO: 265.7 >1000 >1000 >1000 27 SEQ ID NO: 27 25 >1000 >1000 >1000 28 SEQ IDNO: 28 5.6 >1000 >1000 >1000 29 SEQ ID NO: 29 6.5 >1000 >1000 >1000 30SEQ ID NO: 30 7.4 >1000 >1000

The PYY compounds of the inventions all display good Y2 potency, whereasthe potency on the receptors Y1, Y4 and Y5 is strongly reduced.

Example 32 Y1, Y2, Y4 and Y5 Receptor Subtype Binding

The purpose of this example is to test the in vitro binding of the PYYcompounds to the Y1, Y2, Y4 and Y5 receptor subtypes, respectively. Thereceptor binding affinity is a measure of affinity of a compound for thehuman Y1, Y2, Y4 and Y5 receptor subtypes, respectively.

The in vitro binding of the PYY compounds of examples 6-30 weredetermined in a scintillation proximity assay (SPA) as described below.hPYY(3-36) (Example 2, SEQ ID NO:2) was included as a reference.

Scintillation Proximity Assay (SPA)

NPY-Receptor Expressing Cell Lines.

All cells were cultured at +37° C. in a humidified atmosphere with 5%CO₂. BHK-482-8 cells with inducible expression of the human NPY-Y1receptor (P25929, NPY1R_HUMAN, Uniprot) were cultured in Dulbecco'sModified Eagle Medium (DMEM) with 10% Fetal bovine serum (FBS), 1%Penicillin-Streptomycin (P/S), 1 mg/ml G418 antibotic, 1 mg/mlHygromycin B antibiotic and 1% Non-essential amino acids. 1 mM Isopropylβ-D-1-thiogalactopyranoside (IPTG) was added 24 hours prior toharvesting cells for induction of NPY-Y1 receptor expression. CHO-K1cells stably expressing the human NPY-Y2 receptor (P49146, NPY2R_HUMAN,Uniprot) were cultured in DMEM F-12 with 10% FBS, 1% P/S, 150 μg/mlHygromycin B and 10 μg/ml Puromycin antibiotic. CHO-K1 cells stablyexpressing the human NPY-Y4 receptor (P50391, NPY4R_HUMAN, Uniprot) werecultured in DMEM F-12 with 10% FBS, 1% P/S, 10 μg/ml Puromycin. HEK-293cells stably expressing the human NPY-Y5 receptor (Q15761, NPY5R_HUMAN,Uniprot) were cultured in DMEM F-12 medium containing 10% FBS, 1%Penicillin-Streptomycin, 250 μg/ml G418 and 1 μg/ml puromycin.

Membrane Preparation.

Cultured cells were detached mechanically by scraping and washed in icecold PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.47 mM KH₂PO₄ pHadjusted to 7.4) and transferred to tubes and centrifuged for 5 min at1000 g at +4° C. Pellets were resuspended in ice cold homogenizationbuffer; Y1: 20 mM Hepes, 10 mM EDTA, with 2 complete EDTA-free proteaseinhibitor cocktail tablets/50 ml (Roche, Mannheim, Germany) pH 7.4); Y2,Y4: 20 mM Hepes, 5 mM MgCl₂, 1 mg/ml Bacitracin, pH 7.1; Y5: 10 mM NaCl,20 mM Hepes, 0.22 mM KH₂PO₄, 1.26 mM CaCl₂, 0.81 mM MgSO₄, pH 7.4 andthen homogenized for 30 seconds using a tissue homogenizer at mediumspeed. The homogenate was centrifuged at 35000 g using anultracentrifuge for 10 minutes at +4° C. and the supernatant wasdiscarded and fresh homogenization buffer added. Homogenization of thepellet was repeated a total of three times. The final pellet wasresuspended in a few millilitres of homogenization buffer and proteinconcentration was determined using the Bradford method and measured at595 nm in a microplate reader. Protein concentration were adjusted to 1mg/ml and transferred to cryotubes and stored at −80° C. 250 mM sucrosewas added to Y5 membranes prior to freezing.

Assay.

Human NPY-Y receptor SPA binding assay were performed in white 96-wellplates in a total volume of 200p1 per well. Wheat germ agglutinin coatedbeads containing scintillation liquid (PerkinElmer, Waltham, Mass., USA)were reconstituted in binding buffer; Y1, Y2: 50 mM Hepes, 1 mM CaCl₂, 5mM MgCl₂, 0.02% tween 20, 0.25% ovalbumin pH 7.4; Y4, Y5: 20 mM Hepes,10 mM NaCl, 0.22 mM KH₂PO₄, 1.26 mM CaCl₂, 0.81 mM MgSO₄, 0.1%bacitracin and 0.25% ovalbumin pH 7.4 and mixed with membranepreparation to give final concentration of 1 mg beads and 3 μg of Y1membranes/well, 3 μg of Y2 membranes/well, 1 μg of Y4 membranes/well or20 μg of Y5 membranes/well. 50000 cpm per well of radio ligand human[¹²⁵I]-PYY was added corresponding to a concentration of 100 μM in Y1,Y2 and Y5 binding assays. 50000 cpm per well of radio ligand human[¹²⁵1]-Pancreatic Polypeptide (PP) corresponding to a concentration of100 μM was used in Y4 binding assay.

Freeze dried analogues were dissolved in 80% dimethyl sulfoxide (DMSO),19% H₂O and 1% acetic acid (CH₃COOH) to stock solutions of 2000 μM(Y1,Y4 and Y5) and 200 μM (Y2) and serial dilutions (1:10) wereperformed in binding buffer to final concentrations ranging from 10000nM to 1 μM in the Y1, Y4 and Y5 assays and 1000 nM to 0.1 μM in the Y2assay. Plates were sealed and incubated at +25° C. for 2 hours in aplate shaker set at 400 rpm and thereafter centrifuged at 1500 rpm for10 minutes prior to reading of luminescence on a microplatescintillation and luminescence counter. Y1 SPA plates were let to standin room temperature for 16 hours prior to reading. Displacement ofradioligand was measured as reduction in luminescence and IC₅₀ valueswere calculated by nonlinear regression analysis of sigmoidaldose-response curves. Ki values for binding affinity were acquired bythe Cheng-Prusoff equation (Ki=IC50/(1+[L]/Kd) including receptorspecific Kd values (Y1=0.556; Y2=0.275; Y4=0.111; Y5=0.345), radioligandconcentration and IC50 values.

The PYY compounds of the invention all display good Y2 binding while thebinding affinity on the receptors Y1, Y4 and Y5 is strongly reduced.

TABLE 2 Y receptor binding affinity Y2 Y1 Y4 Y5 Example Compound Ki (nM)Ki (nM) Ki (nM) Ki (nM)  2 hPYY(3-36) 0.25 46 83 4.5 SEQ ID NO: 2  6 SEQID NO: 6 1.7 3683.30 5444  7 SEQ ID NO: 7 1.6 6884.67 3555 658  8 SEQ IDNO: 8 2.8 >10000 5743 778  9 SEQ ID NO: 9 0.60 >10000 1449 576 16 SEQ IDNO: 16 3.6 8819.50 >10000 974 17 SEQ ID NO: 17 2.7 5373.00 5559 3801 18SEQ ID NO: 18 13 >10000 >10000 1875 19 SEQ ID NO: 19 7.5 >10000 >100001405 20 SEQ ID NO: 20 3.1 >10000 >10000 2636 21 SEQ ID NO: 215.8 >10000 >10000 1727 22 SEQ ID NO: 22 5.1 >10000 >10000 434 23 SEQ IDNO: 23 9.5 >10000 >10000 972 24 SEQ ID NO: 24 27 >10000 >10000 4839 25SEQ ID NO: 25 7.1 >10000 >10000 4930 26 SEQ ID NO: 26 5.5 >10000 >100002757 27 SEQ ID NO: 27 31 >10000 >10000 5915 28 SEQ ID NO: 287.8 >10000 >10000 2487 29 SEQ ID NO: 29 9.1 >10000 9636 1828 30 SEQ IDNO: 30 15 >10000 >10000 2684

Example 33 Pharmacokinetic Study in Minipigs

The purpose of this study is to determine the half-life in vivo of thePYY compounds after i.v. administration to minipigs, i.e. theprolongation of their time in the body and thereby their time of action.This is done in a pharmacokinetic (PK) study, where the terminalhalf-life of the derivative in question is determined. By terminalhalf-life is generally meant the period of time it takes to halve acertain plasma concentration, measured after the initial distributionphase.

In Vivo Studies on Pharmacokinetic Evaluation in Göttingen Minipigsafter Intravenous Administration.

Animals. Göttingen minipigs female, 15-25 kg, purchased from EllegaardMinipigs, Denmark. The animals were housed in the Animal Unit, NovoNordisk A/S and were kept and handled according to normal procedure inthe Animal Unit.

After minimum 2 weeks of acclimatization two permanent central venouscatheters were implemented in vena cava caudalis in each animal. Aftersurgery the animals were in their normal individual pens during thepharmacokinetic experiments.

Body Weight.

The animals were weighed weekly. The animals were fasted on the morningprior to dosing but had ad libitum access to water; food was suppliedduring dosing.

Administration of Peptides and Dosing Solutions.

Intravenous injections were given through the central short catheter,which was flushed with min 10 ml of sterile saline post administration.The test substance was dosed at 15 nmol/kg, n=3, in a volume of 0.05ml/kg. Buffer: 50 mM sodium phosphate, 70 mM sodium chloride, 0.05%tween 80, pH 7.4 or 20 mM HEPES, 2.2% glycerol, 0.05% Polysorbate 80, pH6.5.

Blood Samples and Analysis.

Blood samples were taken through the central catheter according to thefollowing schedule: Predose, 5, 15, 30, 45 min, 1 h, 1.5 h, 2 h, 3 h, 4h, 6 h, 8 h, 10 h, 24 h, 48 h, 72 h, 96 h, 120 h, 168 h, 192 h, 216 h,240 h, 264 h and 288 h. On day 1 the catheters are coupled to extensiontubes, which will be removed at the end of day 1. Samples (0.8 ml) weretaken through the catheter. Blood was collected in test tubes containingEDTA buffer (8 mM) and 50 μl Val-Pyr buffer (Stabilization buffercontaining 3.097 g K3EDTA dissolved in 50 ml Trasylol and 0.5 ml 20 mMVal-Pyr was added. The pH was regulated to 7.4) After each blood samplethe catheter was flushed with min 5 ml of sterile 0.9% NaCl and 10 IE/mlheparin. Aseptic technique was demanded to avoid bacterial growth in thecatheter that increases the risk of clot formation in the catheter.Samples were kept on wet ice until centrifugation (10 min, 4° C., 1942g). Afterwards, plasma (min. 200 μl) was transferred immediately toMicronic tubes and kept at −20° C. until analysis. The plasma sampleswere analysed by LC/MS as described below.

Data and Results.

Plasma concentration-time profiles was analysed by a non-compartmentalpharmacokinetics analysis using Phoenix (Pharsight Inc., Mountain View,Calif., USA). Calculations were performed using individualconcentration-time values from each animal.

Sample Analysis

Quantitative Assay for Plasma Samples.

The test substances were assayed in plasma by Turbulent FlowChromatography coupled to Liquid Chromatography with subsequent MassSpectrometric Detection (TFC/LC/MS). The selectivity of the methodallowed various compounds to be quantitated in one sample, e.g. cassettedosing of four compounds per animal. The concentrations of the testsubstance in unknown samples were calculated using the peak area as afunction of amount. Calibration graphs based on plasma samples spikedwith the analyte were constructed by regression analysis. Typicaldynamic range for the assay was 1-2,000 nmol/I. The method performancewas assured by co-assaying quality control (QC) samples in duplicate atthree concentration levels. Stock and working solutions of analytes wereprepared in plasma and incubated by 37° C. for 1 hour.

Sample Preparation.

40.0 μl EDTA-plasma was added 160 μl 50% methanol, 1% formic acid, thenvortexed and centrifuged at 14300 rpm (16457 g) at 4° C. for 20 minutes.The supernatant was transferred to a 96 well plate, (the plates havebeen preincubated with 0.4% BSA, 37° C. for 1/2 hour). Injection volumewas 25 μl.

For sample clean up a TurboFlow Cyclone column (0.5×50 mm) both fromThermo Scientific, Franklin, Mass., USA, was used and the LC separationwas done either on an Onyx C18 column (2.0×50 mm) from Phenomenex,Torrance, Calif., USA. Eluents were isocratic and gradient combinationsof methanol, acetonitril, Milli-Q water and formic acid. Selectivedetection was done by mass spectrometry operated in positive modeionisation.

Data Handling.

Plasma concentration-time profiles was analysed by a non-compartmentalpharmacokinetics analysis using Phoenix (Pharsight Inc., Mountain View,Calif., USA). Calculations were performed using individualconcentration-time values from each animal.

TABLE 3 Half-life (t½) t½ Example Compound (hours)  2 hPYY(3-36) 0.3 SEQID NO: 2  7 SEQ ID NO: 7 0.2  8 SEQ ID NO: 8 2.2 11 SEQ ID NO: 11 0.7 16SEQ ID NO: 16 77 17 SEQ ID NO: 17 93 18 SEQ ID NO: 18 58 19 SEQ ID NO:19 62 20 SEQ ID NO: 20 70 22 SEQ ID NO: 22 56 25 SEQ ID NO: 25 85 28 SEQID NO: 28 61 30 SEQ ID NO: 30 66

The tested PYY derivatives of the invention have very long half-lives ascompared to the half-life of hPYY(3-36) and underivatised hPYY(3-36)analogues.

Example 34 Pharmacodynamic Studies in Db/Db Mice

In order to determine the in vivo effects of the PYY compounds on bloodglucose and food intake in db/db mice, the compounds were dosed to db/dbmice and the effects on blood glucose and food intake were measured asdescribed below.

Male db/db mice are housed in a normal daily rhythm (6 μm to 6 am darkcycle) and provided ad libitum access to Altromin diet. At 11-13 weeksof age the mice are matched for blood glucose as well as body weight anddivided into matching groups of 9 mice and housed 3 per cage. Mice aredosed subcutaneously with the indicated compound or vehicle (50 mMNa2HPO4, pH 7.4, 70 mM NaCl, 0.05% Tween 80) at a volume of 2.5 ml/kg atthe indicated doses at 4 μm (time=0) and in some experiments a secondinjection was given at time=23 hours. Blood glucose and food intake aremeasured at the indicated time points post injection, e.g at 4 h, 16 h,23 h and 40 h post injection. Blood samples for blood glucose are takenfrom the tail vein, into a 5 μl heparin coated capillary tube which isplaced in an eppendorf tube with Biosen® system solution (250 μl). Thesamples are analysed on a Biosen® instrument immediately.

Blood glucose (BG) measurements are reported as mean±SEM of vehicleadjusted % BG relative to pre-treatment and calculated as follows:

100−[% BG(vehicle,average)−% BG] where, %BG=100*[BG(time=t)/BG(pre-treatment)]

and % BG(vehicle,average)=average of % BG values for the vehicle groupat time=t relative to vehicle pre-treatment.

Food intake is reported as mean±SEM food intake per cage as a percentageof average food intake of the vehicle group for the indicated interval.

TABLE 4 Effect on blood glucose in db/db mice Dose % change in bloodglucose Example Compound (μmol/kg) 4 h 16 h 23 h 40 h 16 SEQ ID NO: 16 159 73 76 78 17 SEQ ID NO: 17 0.3 79 94 93 96 22 SEQ ID NO: 22 1 69 76 7980 30 SEQ ID NO: 30 1 — 71 63 — 28 SEQ ID NO: 28 1 — 60 53 — 29 SEQ IDNO: 29 1 — 70 61 — 20 SEQ ID NO: 20 1 — 72 57 — 25 SEQ ID NO: 25 1 — 7576 —

TABLE 5 Effect on food intake in db/db mice Dose % relative food intakeExample Compound (μmol/kg) 4 h 16 h 23 h 40 h 16 SEQ ID NO: 16 1 31 2528 38 17 SEQ ID NO: 17 0.3 30 55 57 62 22 SEQ ID NO: 22 1 54 31 36 45 30SEQ ID NO: 30 1 — 29 27 — 28 SEQ ID NO: 28 1 — 20 26 — 29 SEQ ID NO: 291 — 19 22 — 20 SEQ ID NO: 20 1 — 55 105 — 25 SEQ ID NO: 25 1 — 37 41 —

These data strongly support the blood glucose lowering effect and theinhibition of food intake of the PYY compounds of the invention.

While certain features of the invention have been illustrated anddescribed herein, many modifications, substitutions, changes, andequivalents will now occur to those of ordinary skill in the art. It is,therefore, to be understood that the appended claims are intended tocover all such modifications and changes as fall within the true spiritof the invention.

1. A PYY compound comprising L-beta-homoarginine at a positioncorresponding to position 35 of hPYY(1-36) (SEQ ID NO:1), and apharmaceutically acceptable salt thereof.
 2. A PYY compound according toclaim 1, wherein the PYY compound has a maximum of 10 amino acidmodifications as compared to hPYY(3-36).
 3. A PYY compound according toclaim 1, further comprising tryptophan at a position corresponding toposition 30 of hPYY(1-36) (SEQ ID NO: 1).
 4. A PYY compound according toclaim 1, wherein the positions corresponding to positions 1-3 ofhPYY(1-36) (SEQ ID NO: 1) are absent, and wherein the PYY compoundfurther comprises an N-terminal substituent, wherein the N-terminalsubstituent is an alkoxy group comprising up to 12 carbon atoms.
 5. APYY compound according to claim 4, wherein the N-terminal substituent is3-methylbutanoyl.
 6. A PYY compound according to claim 1, furthercomprising a Lysine at a position corresponding to position 7 ofhPYY(1-36) (SEQ ID NO: 1) and a modifying group attached to the epsilonamino group of the Lysine residue in position 7, wherein said modifyinggroup is defined by A-B-C-, wherein A- comprises a carboxylic acid or asulfonic acid.
 7. A PYY compound according to claim 6, wherein A- isselected from

wherein a is an integer from 12 to 19, and b is an integer from 10 to16, and wherein * denotes the attachment point to -B-.
 8. A PYY compoundaccording to claim 6, wherein B- is selected from

wherein c is 1 or 2; and d is 1 or 2; and wherein *** denotes theattachment point to A-, and ** denotes the attachment point to -C-.
 9. APYY compound according to claim 8, wherein B- is

wherein c is 1 or 2; and wherein *** denotes the attachment point to A-,and ** denotes the attachment point to -C-.
 10. A PYY compound accordingto claim 6, wherein -C- is

wherein e is an integer in the range of 1-5, f is an integer in therange of 1-5, g is an integer in the range of 2 to 6, and wherein ****denotes the attachment point to -B-, and ***** denotes the attachmentpoint to the epsilon amino group of the Lysine residue in the positioncorresponding to position 7 of hPYY(1-36).
 11. A PYY compound accordingto claim 10, wherein e and f are each
 1. 12. A PYY compound according toclaim 10, wherein g is selected from 2, 4 or
 6. 13. A PYY compoundaccording to claim 1, selected from the following:


14. (canceled)
 15. A method of treating all forms of diabetes andrelated diseases, eating disorders, diabetic complications,cardiovascular diseases and/or sleep apnoea; and/or for improving lipidparameters, improving β-cell function, and/or for delaying or preventingdiabetic disease progression in a patient requiring such treatmentcomprising administering a pharmaceutically effective amount of apharmaceutical composition comprising the PYY compound according toclaim 1.